*Target (protein/gene name): Succinate dehydrogenase flavoprotein, putative *NCBI Gene # or RefSeq#: XM_804331.1 *Protein ID (NP or XP #) or Wolbachia#: XP_809424.1 *Organism (including strain):Trypanosoma cruzi http://www.ncbi.nlm.nih.gov/nuccore/XM_804331.1 *Background/Disease Information (sort of like the Intro to your Mini Research Write up): Trypanosoma cruzi is a flagellate protozoan that is the cause of Chagas disease, also known as American trypanosomiasis. This disease is characterized as a tropical parasitic disease that is contracted when infected feces from organisms in the family Reduviidae enters the bloodstream. This protozoan can cause death in infected individuals by causing heart failure and disease of the internal organs. There is currently no treatment or cure for chronic forms of infection.
Essentiality of this protein: The protein shows a significant decrease in fitness for T. brucei when inhibited, which means there is probably a similar effect in T. cruzi. Complex of proteins?: Druggable Target: This has a druggability of .8, on a 0 to 1 scale.
*EC#: 1.3.5.1 Link to BRENDA EC# page: --Show screenshot of BRENDA enzyme mechanism schematic Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Colorimetric-continuous assay using iodonitrotetrazolium chloride.
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
Figure1. 1YQ3 homology model for putative protein structure. Labeled by element with carbons green, nitrogen blue, oxygen red, and waters shown as red X
List cost and quantity of substrate reagents and supplier Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 1YQ3
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 98%
---- Max % Identities: 60%
---- % Positives 76%
---- Chain used for homology: Chain A
Current Inhibitors: The structure is putative, but known succinate dehydronase inhibitors are carboxin, thenoyltrifluoroacetone, malate, and oxiloacetate. Expression Information (has it been expressed in bacterial cells): It has been expressed in BL21(DE3)
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MLRRIFARFSPKLSKAYPMIDHTFDCVVVGAGGSGLRAAMGVAASGYDVACISKLYPSRS
HTIAAQGGINAALANCEEDDWRWHVYDTVKGSDWLGDQDAIQYMCQEAPCVVSELESMGL
PFLRTKDGFIYQRAFGGQSIHFGGKQARRTCAASDRTGHAMLHTLYGQSFQYGVNFFNEY
YCLDLIVEDGCCRGVVAMSIDDGTIHRFRSKYTILATGGYGRCWFTTTSAKSCTGDGTAM
VARAGLPAEDMEFVQFHPTGIYGPGVLITEGARGEGGYLVNSEGERFMERYAPKAKDLAS
RDVVSRAITLEVLAGRGCGPKKDHVLLQLHHLPPEQLHEKLPGISESAHIFAGVDVTKES
IPIVPTVHYNMGGIPTLWTGEVVSPRNGDDDAIVPGLLAAGECACASVHGANRLGANSLL
DIVVFGKSCANTVIFNLTKEGRNQPELRPDAGESSIADLDGILHNKGDIPVARIRERMKE
TMALYAAVFRTEESMLKGQGIIEECYRDYSHVFVHDKSPVWNSNLIEALELRNLLTNALL
TITGAAVRRESRGAHARDDYPERDDHNWMKHTLAYLDDKNGKARLAYRRVHNEMLTNELE
SIPPAKRVY http://earth.lab.nig.ac.jp/~dclust/cgi-bin/barley_pub/blast_viewer/showflatdata.cgi?CLNID=45654&CLNLIBID=54&DBID=1#115 *length of your protein in Amino Acids 609aa Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website 66903.9 grams/mole Molar Extinction coefficient of your protein at 280 nm wavelength: 73770 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. *CDS Gene Sequence (paste as text only):
atgcttcgcc gcatatttgc ccgcttctcc ccgaagctga gtaaggcgta cccgatgatt
1801 agcatccctc ctgccaagcg tgtttactag *GC% Content for gene: *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Because the structure is putative, the above information is not available yet. http://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_71414659 Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
*NCBI Gene # or RefSeq#: XM_804331.1
*Protein ID (NP or XP #) or Wolbachia#: XP_809424.1
*Organism (including strain): Trypanosoma cruzi
http://www.ncbi.nlm.nih.gov/nuccore/XM_804331.1
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Trypanosoma cruzi is a flagellate protozoan that is the cause of Chagas disease, also known as American trypanosomiasis. This disease is characterized as a tropical parasitic disease that is contracted when infected feces from organisms in the family Reduviidae enters the bloodstream. This protozoan can cause death in infected individuals by causing heart failure and disease of the internal organs. There is currently no treatment or cure for chronic forms of infection.
Essentiality of this protein: The protein shows a significant decrease in fitness for T. brucei when inhibited, which means there is probably a similar effect in T. cruzi.
Complex of proteins?:
Druggable Target: This has a druggability of .8, on a 0 to 1 scale.
*EC#: 1.3.5.1
Link to BRENDA EC# page:
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Colorimetric-continuous assay using iodonitrotetrazolium chloride.
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.sigmaaldrich.com/catalog/product/sial/i8377?lang=en®ion=US-- link (or citation) to paper that contains assay information:
http://www.ncbi.nlm.nih.gov/pubmed/8214593
List cost and quantity of substrate reagents and supplier
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 1YQ3
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 98%
---- Max % Identities: 60%
---- % Positives 76%
---- Chain used for homology: Chain A
Current Inhibitors: The structure is putative, but known succinate dehydronase inhibitors are carboxin, thenoyltrifluoroacetone, malate, and oxiloacetate.
Expression Information (has it been expressed in bacterial cells): It has been expressed in BL21(DE3)
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MLRRIFARFSPKLSKAYPMIDHTFDCVVVGAGGSGLRAAMGVAASGYDVACISKLYPSRS
HTIAAQGGINAALANCEEDDWRWHVYDTVKGSDWLGDQDAIQYMCQEAPCVVSELESMGL
PFLRTKDGFIYQRAFGGQSIHFGGKQARRTCAASDRTGHAMLHTLYGQSFQYGVNFFNEY
YCLDLIVEDGCCRGVVAMSIDDGTIHRFRSKYTILATGGYGRCWFTTTSAKSCTGDGTAM
VARAGLPAEDMEFVQFHPTGIYGPGVLITEGARGEGGYLVNSEGERFMERYAPKAKDLAS
RDVVSRAITLEVLAGRGCGPKKDHVLLQLHHLPPEQLHEKLPGISESAHIFAGVDVTKES
IPIVPTVHYNMGGIPTLWTGEVVSPRNGDDDAIVPGLLAAGECACASVHGANRLGANSLL
DIVVFGKSCANTVIFNLTKEGRNQPELRPDAGESSIADLDGILHNKGDIPVARIRERMKE
TMALYAAVFRTEESMLKGQGIIEECYRDYSHVFVHDKSPVWNSNLIEALELRNLLTNALL
TITGAAVRRESRGAHARDDYPERDDHNWMKHTLAYLDDKNGKARLAYRRVHNEMLTNELE
SIPPAKRVY
http://earth.lab.nig.ac.jp/~dclust/cgi-bin/barley_pub/blast_viewer/showflatdata.cgi?CLNID=45654&CLNLIBID=54&DBID=1#115
*length of your protein in Amino Acids 609aa
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website 66903.9 grams/mole
Molar Extinction coefficient of your protein at 280 nm wavelength: 73770
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
atgcttcgcc gcatatttgc ccgcttctcc ccgaagctga gtaaggcgta cccgatgatt
61 gaccacacct ttgactgtgt ggtggttggt gccggtggtt cagggcttcg tgctgcgatg
121 ggcgttgccg cgtccggcta cgacgtggca tgcatctcca aactctatcc atcaaggtca
181 cacaccattg ctgcccaggg cggcatcaac gcagcacttg ccaactgtga ggaggacgac
241 tggcgttggc atgtgtacga taccgtgaag ggaagtgact ggctggggga ccaggatgca
301 attcagtaca tgtgccaaga agctccgtgt gtggtctcag aactagagag catgggactt
361 cccttcttgc gtacaaaaga tggatttatt tatcaacgtg catttggcgg ccaatctatt
421 cactttggtg gaaaacaggc gcgccggacg tgtgccgcgt cggaccgcac gggacacgca
481 atgctccaca cactctatgg gcaatcgttt cagtacggcg tgaacttttt taacgaatat
541 tattgcttag acctgattgt agaagatgga tgctgccgtg gagtggttgc catgagcatc
601 gatgatggca caattcatcg cttccgatcc aagtacacta ttcttgccac cggtggttac
661 ggccgctgct ggtttacaac aacaagcgcc aagtcttgta ccggagacgg gacggcaatg
721 gtggcacgtg cagggctgcc ggcggaagac atggaatttg tgcagttcca cccaacggga
781 atttacggtc ctggtgtgct gattaccgag ggggcccgtg gcgagggtgg ttacctcgtc
841 aacagtgaag gggagcgctt catggagcga tacgccccca aggcaaagga tttggcgtct
901 cgtgatgtgg tgtcacgcgc cattacgttg gaggttcttg caggacgtgg ctgtggcccc
961 aagaaggatc acgtcctgct gcaacttcat catctcccac ccgagcaact acatgagaag
1021 cttcccggta tctccgagag cgcacatatt tttgcgggtg tggacgtgac gaaggagtcc
1081 atccctatcg tgccgacggt gcactacaac atgggaggca tcccaacatt gtggacgggc
1141 gaggtagtca gtccccggaa tggcgatgac gatgcgatcg tcccaggtct tttggcggcg
1201 ggcgagtgtg cgtgcgcgag tgtgcatggg gcaaatcgtc tgggtgccaa ctccctcctc
1261 gacattgttg tttttggcaa atcatgtgcg aacaccgtca tatttaacct gacgaaggaa
1321 ggacgcaatc agccggagct gcggcccgat gccggggagt cgtccattgc cgatcttgac
1381 ggcattctgc acaacaaggg ggacatccct gtcgcacgca tccgtgagcg catgaaggag
1441 acgatggcgc tctacgcagc cgtgtttcgc actgaggaga gcatgctcaa gggacagggg
1501 atcattgaag aatgttaccg ggattactcc catgtgtttg tccacgacaa gtccccggtg
1561 tggaacagca accttattga ggcgctcgag ctgcgtaatt tgctcacaaa cgcgcttttg
1621 accatcactg gagctgccgt gcggcgggag tctcgtgggg cacatgcacg cgacgactac
1681 ccggagcgag acgaccacaa ctggatgaag catacacttg cgtacctcga tgacaaaaac
1741 ggtaaagccc gtcttgcgta ccgccgagta cacaacgaaa tgctaacaaa tgaacttgaa
1801 agcatccctc ctgccaagcg tgtttactag
*GC% Content for gene:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Because the structure is putative, the above information is not available yet.
http://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_71414659
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):